A. Field of the Invention
The present invention relates to the preparation and use of electrophoresis gels. The invention makes use of ultra-thin slab gels for the separation of macromolecules.
B. Description of the Art
Electrophoresis is the process of separating molecules on the basis of the molecule's migration in an electric field. In an electric field, a molecule will migrate towards the pole that carries a charge opposite to the charge carried by the molecule. The charge carried by a molecule often depends upon the pH of the medium in which the molecule is migrating. A common electrophoretic procedure is to set up solutions at different pH at each end of an electric field. At a certain pH, the isoelectric point of a molecule is obtained and the molecule carries no net charge. Therefore, as the molecule crosses the pH gradient, the molecule reaches an isoelectric point and is immobile in the electric field. This procedure separates molecules according to their different isoelectric points.
Electrophoresis in a polymeric gel, such as polyacrylamide or agarose, adds two advantages to an electrophoretic system. First, the polymeric gel stabilizes the electrophoretic system against convective disturbances. Second, the polymeric gel provides a porous passageway through which the molecules travel. Since larger molecules will travel more slowly through the passageways than smaller molecules, use of a polymeric gel permits the separation of molecules by either molecular size or isoelectric point.
Electrophoresis in a polymeric gel is also often used to separate molecules only by molecular size. Some groups of molecules, such as RNA and DNA molecules, all have the same electrophoretic mobility in free solution. These groups of molecules when moved through a polymeric gel by an electric field will segregate on the basis of molecular size. Thus nucleic acids and other groups of molecules with similar isoelectric points will migrate through the gel to be segregated solely on the basis of molecular size.
A polymeric gel electrophoresis system is typically set up in the following way: A gel-forming solution is allowed to polymerize between two glass plates that are held apart on two sides by spacers. These spacers determine the thickness of the gel. Sample wells are formed by inserting a comb-shaped mold into the liquid between the glass plates at one end and allowing the liquid to polymerize around the mold. The top and bottom of the polymerized gel are in electrical contact with two buffer reservoirs. Macromolecule samples are loaded into the sample wells. An electric field is set up across the gel, and the molecules begin to separate according to their size.
The size-sorted molecules can be visualized in several ways. After electrophoresis, the gels can be bathed in a DNA-specific or protein-specific stain which renders the groups of size-sorted molecules visible to the eye. For greater sensitivity, the molecules can be radioactively labelled and the gel exposed to X-ray film. The developed X-ray film will indicate the migration positions of the labelled molecules.
Both vertical and horizontal assemblies are routinely used in gel electrophoresis. The molecules can also be detected during electrophoresis, either by means of their intrinsic absorptive or fluorescent properties, or by labelling them with a detectable chromophore or fluorophore, or by other detection methods known in the art. In a vertical apparatus, the sample wells are formed in the same plane as the gel and are loaded vertically. The wells can be as deep and wide as needed, but the thickness of the well is limited by the thickness of the gel. If ultra-thin (&lt;0.15 mm) gels are cast, loading the sample can be troublesome.
Ultra-thin electrophoretic gels are useful because they may be electrophoresed at a higher voltage. Therefore, the electrophoretic run is faster. Ultra-thin often gels yield higher resolution. Because of their thinness, the gels are fixed for autoradiography quickly and easily.
Sample wells in a horizontal apparatus are typically formed into the thickness of the gel and are loaded vertically. The wells may be of any desired thickness and, hence, are easier to load than the wells formed in a vertical apparatus. The depth of the wells is limited by the thickness of the horizontal gel.
The use of horizontal assemblies is known in the art. For example, Hurd, et al., U.S. Pat. Nos. 4,909,977 and 4,795,591, claim such a horizontal apparatus. The sample wells of the Hurd apparatus are formed by a comb-shaped mold at the extreme end of the slab gel. The comb is placed into a slot formed between the side of the bottom tray and the edge of the top tray.
Several problems are experienced by prior horizontal electrophoresis assemblies. The comb is held in place by the pressure of the top tray, but unless the comb is held very tightly, non-uniform sample wells with exogenous gel material will be formed. Additionally, the sample well geometry is such that the electric field "turns a corner" in the area of the sample well. This non-uniform electric field geometry will cause artifactual migration in this area of the gel and may cause the samples to electrophorese aberrantly. Finally, the apparatus is suitable to cast gels of 0.15-0.3 mm thick, so there is no teaching of the special problems of ultra-thin sample well formation.
The usefulness of electrophoresis depends on the sharp resolution of sample separation. This sharp resolution depends, in part, on the manner in which the macromolecular sample migrates from the sample well. Both the sample well and the electric field influence the migration of the macromolecules. Ideally, the sample wells would have a uniform, sharply defined size and have no extraneous pieces of polymerized gel that would interfere with sample migration. Uniform well size is necessary because the separation of molecules is often compared between samples that are electrophoresed side-by-side. Extraneous material in the sample well will cause impeded migration for part of the sample. Non-uniform sample migration greatly hinders high resolution molecular separation.
The placement of the electric field relative to the sample well is important because a non-uniform field can create artifactual results. Ideally, the electric field experienced by the loaded samples would be in a plane parallel to the gel, even when the sample is in the sample well. Then the molecules would experience a uniform electric field during their entire electrophoretic separation.
What is needed is an apparatus and method for casting ultra-thin gels having sample wells with a sharply defined, uniform geometry and having sample wells positioned so that the electric field passing through the sample well is parallel to the plane of the slab gel.